This present invention relates generally to microbubbles and, more particularly, to naturally occurring gas vesicles. Specifically, the present invention relates to methods for delivering or removing a gas to or from a site using these naturally occurring gas vesicles as well as methods for harvesting, modifying, and exploiting the same for various applications. Because the methods of the present invention also entail modifying the naturally occurring gas vesicles, the present invention further relates to semi-synthetic gas vesicles. Compositions of matter comprising these gas vesicles are also disclosed.
Microbubbles, encompassing both natural and synthetic gas-filled microcavities, are well known in the art. For example, gas-filled microcavities have been employed for enhanced oil recovery, as contrast agents in diagnostic ultrasound, as reagents in in situ bioremediation of contaminated ground water, and as flotation devices for the separation of minerals.
Microbubbles used heretofore in the art have been synthetic in nature. That is, microbubbles have been produced by methods such as passing air through a surfactant solution. One known technique provides a rapid flow of a dilute surfactant solution through a venturi throat through which gas is emitted to generate the surfactant-stabilized microbubbles. Another example includes employing a triple-barreled jet head that allows for the simultaneous development of an alginate drop and injection of an air bubble inside the drop. Depending on the production type, these microbubbles have been referred to as microballoons, colloidal gas aphrons, micro gas dispersions, and microfoams.
Although synthetically produced microbubbles are well known in the art, they have several shortcomings. Namely, synthetically produced microbubbles lack consistency of size, have poor stability and mechanical strength, and are often biologically incompatible.
Naturally occurring microbubbles, such as gas vesicles, are also known. Many organisms produce and/or employ microbubbles for various biological functions. Specifically, ecological studies show that many microorganisms living in aquatic systems utilize microbubbles as buoyancy devices. Their importance in providing buoyancy for planktonic cyanobacteria and helping them perform vertical migration in lakes and other aquatic systems has been widely recognized. Additionally, they are postulated to play a role in light shielding, as well as providing the cell with the ability to alter its configuration to increase cell surface area as a function of volume.
Among the difficulties in utilizing naturally occurring microbubbles for commercial purposes is the fact that it is difficult to harvest them. For example, in typical industrial microbial fermentations, cells are collected by either filtration or centrifugation. Filtration involves large pressure gradients or mechanical forces that tend to collapse the gas vesicles, and centrifugation is inefficient because the vesicle-bearing cells often have densities very close to that of water. Further, where centrifugal force is strong enough to achieve efficient cell collection, such forces often destroy the gas vesicles. It is also difficult to sterilize them so that they can be kept stable against microbial and enzymatic attack.
Biological systems often produce gaseous compounds as by-products of their metabolism. When these compounds accumulate, they can inhibit the growth, product synthesis and even survival of the biological system. A common example of such a gaseous compound is carbon dioxide. If it is not removed effectively, carbon dioxide accumulation can negatively affect plant cell cultures, insect cell cultures, animal cell cultures, and microbial fermentations. Furthermore, the low shear requirements of many types of cell culture lead to poor gas-liquid interfacial transfer and, consequently, potential accumulation of inhibitory or toxic gaseous metabolic by-products.
Thus, there is a need in the art to overcome the shortcomings of synthetically produced microbubbles. Further, there is a need in the art to overcome the difficulties in harvesting naturally occurring microbubbles and utilizing such microbubbles for commercial purposes in lieu of synthetic microbubbles. Still further, there is a need in the art to overcome the difficulties associated with the removal of gaseous metabolic byproducts from biological systems.
It is therefore an object of the present invention to provide purified gas vesicles for use in delivery or removal of a gas to or from a site.
It is another object of the present invention to provide semi-synthetic microbubbles with improved strength or improved stability.
It is yet another object of the present invention to provide crosslinked gas vesicles in a sterile suspension.
It is a further object of the present invention to provide a method for harvesting naturally occurring microbubbles.
It is another object of the present invention to provide a method for modifying naturally occurring microbubbles to produce semi-synthetic microbubbles.
It is still another object to provide a method for delivering or removing a gas using semi-synthetic microbubbles.
It is yet another object to provide a method for delivering oxygen or other desirable gaseous compounds or for removing undesirable gaseous compounds from a cell or tissue culture via semi-synthetic microbubbles.
At least one or more of the foregoing objects, together with the advantages thereof over the known art relating to microbubbles, which shall become apparent from the specification which follows, are accomplished by the invention as hereinafter described.
In general, the present invention provides a method for delivering a gas to a site, or alternatively, for removing a gas from a site, comprising placing cells having gas vesicles under conditions that induce the cells to float to a surface of an aqueous medium, harvesting the cells from the surface of the medium, lysing the cells, separating the gas vesicles from the lysed cells, and crosslinking the gas vesicles with a crosslinking agent. In removing a gas, the method requires the additional step of loading the gas vesicles with a gas that has a higher partial pressure for the gas to be delivered and/or that has a lower partial pressure of the compound or compounds to be removed from the site, and placing the gas vesicles such that the gas to be delivered partitions from the vesicles and the gas to be removed partitions into the vesicles.
The present invention also provides a method of harvesting gas-vesicle containing cells comprising placing cells having gas vesicles under conditions that induce the cells to float to a surface of an aqueous medium, and harvesting the cells from the surface of the medium.
The present invention further provides isolated gas vesicles comprising a medium having extracellular gas vesicles, preferably crosslinked, and having improved strength or improved stability.